Juerg H. Beer

Platelet production and platelet destruction: assessing mechanisms of treatment effect in immune thrombocytopenia

This study investigated the immature platelet fraction (IPF) in assessing treatment effects in immune thrombocytopenia (ITP). IPF was measured on the Sysmex XE2100 autoanalyzer. The mean absolute-IPF (A-IPF) was lower for ITP patients than for healthy controls (3.2 vs 7.8 × 10⁹/L, P < .01), whereas IPF percentage was greater (29.2% vs 3.2%, P < .01). All 5 patients with a platelet response to Eltrombopag, a thrombopoietic agent, but none responding to an anti-FcγRIII antibody, had corresponding A-IPF responses. Seven of 7 patients responding to RhoD immuneglobulin (anti-D) and 6 of 8 responding to intravenous immunoglobulin (IVIG) did not have corresponding increases in A-IPF, but 2 with IVIG and 1 with IVIG anti-D did. This supports inhibition of platelet destruction as the primary mechanism of intravenous anti-D and IVIG, although IVIG may also enhance thrombopoiesis. Plasma glycocalicin, released during platelet destruction, normalized as glycocalicin index, was higher in ITP patients than controls (31.36 vs 1.75, P = .001). There was an inverse correlation between glycocalicin index and A-IPF in ITP patients (r² = -0.578, P = .015), demonstrating the relationship between platelet production and destruction. Nonresponders to thrombopoietic agents had increased megakaryocytes but not increased A-IPF, suggesting that antibodies blocked platelet release. In conclusion, A-IPF measures real-time thrombopoiesis, providing insight into mechanisms of treatment effect.

Dietary α-linolenic acid increases the platelet count in ApoE−/− mice by reducing clearance

Previously we reported that dietary intake of alpha-linolenic acid (ALA) reduces atherogenesis and inhibits arterial thrombosis. Here, we analyze the substantial increase in platelet count induced by ALA and the mechanisms of reduced platelet clearance. Eight-week-old male apolipoprotein E knockout (ApoE(-/-)) mice were fed a 0.21g% cholesterol diet complemented by either a high- (7.3g%) or low-ALA (0.03g%) content. Platelet counts doubled after 16 weeks of ALA feeding, whereas the bleeding time remained similar. Plasma glycocalicin and glycocalicin index were reduced, while reticulated platelets, thrombopoietin, and bone marrow megakaryocyte colony-forming units remained unchanged. Platelet contents of liver and spleen were substantially reduced, without affecting macrophage function and number. Glycoprotein Ib (GPIb) shedding, exposure of P-selectin, and activated integrin αIIbβ3 upon activation with thrombin were reduced. Dietary ALA increased the platelet count by reducing platelet clearance in the reticulo-endothelial system. The latter appears to be mediated by reduced cleavage of GPIb by tumor necrosis factor-α-converting enzyme and reduced platelet activation/expression of procoagulant signaling. Ex vivo, there was less adhesion of human platelets to von Willebrand factor under high shear conditions after ALA treatment. Thus, ALA may be a promising tool in transfusion medicine and in high turnover/high activation platelet disorders.

Platelet microparticle suppressing antibody against GP Ibα acts independently of the filamin cleavage and increases protein tyrosine phosphorylation

Our aim was to investigate the role of filamin cleavage and protein tyrosine phosphorylation in shear-stress-induced platelet microparticle formation and of its suppression by the monoclonal antibody (mAb) Ib-23 directed against GPIbα. PPACK-anticoagulated or EDTA-anticoagulated platelet-rich plasma or washed platelets were exposed to high shear stress (5000 s−1 for 5 min) in the presence of antagonists of GP Ibα (mAb Ib-23), of GP IIb/IIIa (abciximab) and their combination. We assessed the generation of microparticle by flow cytometry, the filamin cleavage and the protein tyrosine phosphorylation by western blotting. Microparticle formation decreased by more than 50% with mAb Ib-23 alone (P < 0.01, n = 23) but filamin cleavage was not inhibited. In contrast, abciximab did not change microparticle numbers nor filamin cleavage under the same conditions. However, when combined, the inhibitors against GP Ibα and GP IIb/IIIa decreased microparticle by 70% and the filamin cleavage by 20% (P < 0.05). Focal adhesion kinase and p72Syk phosphorylation was enhanced by mAb Ib-23, whereas treatment with abciximab reduced it. mAb Ib-23 inhibits platelet microparticle induced by high shear stress. The mechanism seems not to depend on filamin cleavage: abciximab allowed for full microparticle formation at similar levels of cleaved filamin, whereas the combined inhibitors reduced it. The effect of mAb Ib-23 on protein tyrosine phosphorylation supports previous data, which correlates microparticle formation with the extent of protein tyrosine dephosphorylation and mirrors the dephosphorylation by protein tyrosine phosphatase (PTP-1B) in platelets of calpain−/− knockout mice.

Ticagrelor, but not clopidogrel, reduces arterial thrombosis via endothelial tissue factor suppression

Aims The P2Y12 antagonist ticagrelor reduces mortality in patients with acute coronary syndrome (ACS), compared with clopidogrel, and the mechanisms underlying this effect are not clearly understood. Arterial thrombosis is the key event in ACS; however, direct vascular effects of either ticagrelor or clopidogrel with focus on arterial thrombosis and its key trigger tissue factor have not been previously investigated. Methods and results Human aortic endothelial cells were treated with ticagrelor or clopidogrel active metabolite (CAM) and stimulated with tumour necrosis factor-alpha (TNF-&agr;); effects on procoagulant tissue factor (TF) expression and activity, its counter-player TF pathway inhibitor (TFPI) and the underlying mechanisms were determined. Further, arterial thrombosis by photochemical injury of the common carotid artery, and TF expression in the murine endothelium were examined in C57BL/6 mice treated with ticagrelor or clopidogrel. Ticagrelor, but not CAM, reduced TNF-&agr;-induced TF expression via proteasomal degradation and TF activity, independently of the P2Y12 receptor and the equilibrative nucleoside transporter 1 (ENT1), an additional target of ticagrelor. In C57BL/6 mice, ticagrelor prolonged time to arterial occlusion, compared with clopidogrel, despite comparable antiplatelet effects. In line with our in vitro results, ticagrelor, but not clopidogrel, reduced TF expression in the endothelium of murine arteries. Conclusion Ticagrelor, unlike clopidogrel, exhibits endothelial-specific antithrombotic properties and blunts arterial thrombus formation. The additional antithrombotic properties displayed by ticagrelor may explain its greater efficacy in reducing thrombotic events in clinical trials. These findings may provide the basis for new indications for ticagrelor.

In response to the comment by Hechler et al.: Amotosalen/UVA pathogen inactivation technology reduces platelet activatability, induces apoptosis and accelerates clearance.

We would like to thank B. Hechler and colleagues for the interest shown in our paper on the effects of the Amotosalen/UVA on platelet function, and we are pleased to take the opportunity to reply to their comments. They note correctly that the results of our study are divergent from their